Cyanamide (NH_2CN) has been industrially produced in large quantities for a long time. We have isolated cyanamide as a plant growth inhibitor from hairy vetch Vicia villosa. Although we excluded the possibility that the cyanamide isolated was an unexpectedly contaminating agrochemical, the details of its biosynthesis remain unknown because this compound has never been considered as a natural product. In this report we demonstrate the de novo production of cyanamide in this legume using a ^<15>N-labeled nitrogen source. The extracts of the seedlings of V. villosa grown with and without (^<15>N)nitrate were purified chromatographically to give cyanamide, respectively. On the basis of the GC/MS analyses, the isotopic ratio ^<15>N/(^<14>N+^<15>N) of the ^<15>N-enriched cyanamide was calculated to be 0.143, while that of the non-enriched cyanamide was 0.0065. We also measured the mass spectrum of dicyandiamide [NH_2C(=NH)NHCN], a dimmer of cyanamide, prepared from the purified cyanamide. The isotopic ratio was 0.138, corresponding well to the result of the GC/MS analysis of cyanamide. From these observations, the incorporation of the nitrogen atom of nitrate into cyanamide has been established. We conclude that cyanamide is de novo biosynthesized in V. villosa.
